Systematic analysis of siRNA and mRNA features impacting fully chemically modified siRNA efficacy

Area of Science:

• Biotechnology
• Molecular Biology
• Pharmacology

Background:

• Chemically modified small interfering RNAs (siRNAs) represent a promising therapeutic strategy for silencing disease-causing genes through messenger RNA (mRNA) degradation.
• The efficacy of siRNAs is influenced by both siRNA-specific characteristics (sequence, modification, structure) and target mRNA properties.

Purpose of the Study:

• To systematically determine the contributions of siRNA sequence, structure, and modification patterns relative to the target mRNA's native context.
• To inform the design of therapeutic siRNAs and promote their broader clinical application.

Main Methods:

• Synthesis of approximately 1260 differentially modified siRNAs.
• Evaluation of siRNA silencing efficiency against therapeutically relevant mRNAs (APP, BACE1, MAPT, SNCA) using reporter-based and native expression assays.

Main Results:

• The siRNA modification pattern, such as 2'-O-methyl content, significantly affects gene silencing efficacy.
• Structural siRNA features, like symmetric versus asymmetric configurations, did not impact efficacy.
• Significant variability in effective siRNA 'hit rates' was observed across different target mRNAs.
• Reporter assays mitigated target-specific differences in hit rates, highlighting the role of native mRNA features.

Conclusions:

• Native mRNA features, including exon usage, polyadenylation site selection, and ribosomal occupancy, partially explain siRNA efficacy variability.
• A framework for optimizing therapeutic siRNA design based on these insights has been proposed.